Analytical Sciences
Online ISSN : 1348-2246
Print ISSN : 0910-6340
ISSN-L : 0910-6340
Original Papers
Protein-based Open Sandwich Immuno-PCR for Sensitive Detection of Small Biomarkers
Sharif HASANJinhua DONGYuko HARAYoshihito MORIZANEFutoshi SHIBASAKIHiroshi UEDA
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JOURNALS FREE ACCESS

2013 Volume 29 Issue 9 Pages 871-876

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Abstract

Open sandwich (OS) immunoassay utilizes antigen-dependent stabilization of an antibody variable region to quantify various antigens, enabling noncompetitive detection of small molecules with a broad working range. For further improvement of its sensitivity, OS Immuno-PCR was attempted with recombinant fusion proteins. The maltose binding protein-fused heavy chain variable region (MBP-VH) of an antibody that recognizes the C-terminal fragment of human osteocalcin (bone Gla protein, BGP), a biomarker for bone-related diseases, was immobilized onto microplate wells, and the antigen together with streptavidin (SA)-fused light chain variable region of the same antibody (SA-VL) was added and incubated. The amount of immobilized SA-VL was quantified by tethered biotinylated DNA, which was used to estimate the amount of antigen by realtime PCR. When BGP C-terminal peptide was detected, the limit of detection was 100 fg/mL, which was superior than that of our previously reported phage-based OS Immuno-PCR. The developed OS Immuno-PCR system will be useful for the detection of small molecule biomarkers for disease prevention.

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© 2013 by The Japan Society for Analytical Chemistry
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